1. Field of the Invention
This invention relates to the field of immunodiagnostics and radioimmunotherapy and, more particularly, to methods for labeling antibodies, fragments thereof and peptides with metal ions.
2. Background of the Invention
Proteins have been labeled with various radiometals and other radioisotopic elements for use in immunodiagnostic and immunotherapeutic procedures. Some radiometals have superior properties for use in these techniques. Technetium-99m is an ideal radionuclide for scintigraphic imaging because of its nuclear properties. It has a single photon energy of 140 KeV, a half-life of about 6 hours, and it is readily available from a .sup.99 Mo-.sup.99m Tc generator.
Two general approaches have been taken to label proteins such as antibodies with radiometals. The first is the direct labeling method by which the radiometal is bound to the protein molecule itself. The second is the indirect labeling method in which a complexing agent is coupled to the protein and the radiometal is attached to the protein via the complexing agent.
Rhodes discloses a method of direct labeling of protein with technetium-99m which involves ligand solid phase exchange. See U.S. Pat. No. 4,305,922. According to the method of Rhodes, pertechnetate is reduced to technetium IV and then applied onto a Sephadex.RTM. column. The reduced technetium-99m binds to the Sephadex.RTM. material. A solution of the protein to be labeled is poured onto the top of the Sephadex column where it is allowed to remain so that ligand exchange occurs. As a result, the technetium-99m is transferred preferentially from the Sephadex.RTM. material to the protein. The protein may be pretreated with stannous chloride (a procedure called "pretinning") to enhance transfer of the radiometal to the protein. See U.S. Pat. No. 4,424,200.
Various attempts have been made to label proteins with radiometals by the indirect approach. In one such approach, a chelating agent such as diethylenetriaminepentaacetic acid (DTPA) is conjugated onto the protein and then the metal ion is labeled onto the chelating agent attached to the protein molecule. For example, Khaw et al., Science 209: 295-297 (1980) discloses antibodies to cardiac myosin labeled with indium-111 via DTPA and use of the labeled antibodies to image for myocardial infarction. See also, Krejcarek et al., Biochem. Biophys. Res. Commun. 77: 581-585 (1977); Childs, R. L. and Hnatowich, D. J., J. Nucl. Med. 26: 293 (1985). In a more recent approach, Fritzberg et al. describe the use of particular diamidodithiol and diaminodithiol groups, as a chelating agents. Fritzberg et al, J. Nucl. Med. 27: 957 (1986); European Patent Application 86100360.6.
Various degrees of success have been achieved with both the direct and indirect methods of labeling proteins with radiometals; however, the labeled product is often unstable in vivo. Also, techniques for purifying the labeled product before use are often required. Methods for labeling proteins can be further complicated when radiometals such as technetium (Tc) or rhenium (Re), desirable because of their nuclear properties and ready availability, are used. Such radiometal are available in oxidized states and must be subjected to reducing conditions before labeling. Since such radiometals have relatively short half-lives, time-consuming labeling procedures are obviously disadvantageous.
Clearly, there are numerous obstacles to a simple and efficient method for radiolabeling proteins, one which can be conveniently, rapidly and reliably performed by a clinician or technician prior to the use of the labeled protein as a therapeutic or diagnostic tool.